Significant progress has been made in the understanding of bone remodeling, especially related to the cell biology of bone resorption, in recent years. Advances in the biology of the osteoclast have been contributed to by work supported through this project. The long-term goals of this renewal application are to provide the basis for precise manipulation of bone resorption in disorders such as osteoporosis and renal osteodystrophy by an in-depth understanding of the biology of osteoclast function. The studies in this proposal analyze signal transduction which results from bone matrix proteins interacting with osteoclasts as they attach to bone surfaces. The bone matrix protein, osteopontin, serves as a ligand for the osteoclast integrin alpha(v)beta(3). Osteopontin stimulates a series of immediate cell signals which result in the formation of the osteoclast sealing zone and the insertion of lysosomal proteins into the specialized area of the plasma membrane termed the "ruffled border". Bone matrix - integrin-based signal transduction involves activation of a complex cascade of tyrosine phosphorylation and post-translational modifications of signalling proteins. A critical component of this cascade is the non-receptor tyrosine kinase, c-src. This was demonstrated by the fact that c-src knockout produced a non-functioning osteoclast which failed to form ruffled borders. The studies in this application will carefully analyze the location of critical c-src actions between the alpha(v)beta(3) integrin and downstream effectors. Studies will also analyze the regulation of the GTP binding proteins responsible for the formation of the sealing zone through stimulation of podosome assembly. The Specific Aims of the project are to: 1) analyze the mechanism of gelsolin-associated phosphatidylinositol 3-hydroxyl kinase stimulation by osteopontin; and 2) analyze the mechanism of osteopontin stimulation of podosome assembly. Direct assays of protein/protein interactions will be utilized along with a cell culture model of c-src knockout. The mechanism of c-src activation through dephosphorylation of tyrosine 527 will be analyzed. Studies in Specific Aim #2 will utilize constitutively active analogues of the rho family of GTP binding proteins, microinjected into osteoclasts, to study their role in osteopontin stimulation of podosome assembly. Specific inhibitors and dominant negative analogues of the proteins will be used to interrupt signalling pathways. It is suggested that the results of the proposed studies will represent significant advances in the cell biology of the osteoclast and provide important leads for pharmacologic product development.